CN109666750B - Multiplex real-time fluorescence PCR detection primer probe set and detection method for identifying streptococcus suis and pasteurella multocida - Google Patents
Multiplex real-time fluorescence PCR detection primer probe set and detection method for identifying streptococcus suis and pasteurella multocida Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biological strain detection, in particular to a multiplex real-time fluorescence PCR detection primer composition for identifying streptococcus suis and pasteurella multocida and a detection method. The method can be used for providing qualitative detection of the streptococcus suis and the pasteurella multocida, can be used for accurate quantification, has high amplification efficiency, high sensitivity, high accuracy, good reproducibility and short detection period, can complete detection within 1.5 hours, can monitor in real time, has high feasibility and application prospect, and provides a scientific and reliable method for identifying pathogenic microorganisms and reducing economic loss of breeding.
Description
Technical Field
The invention relates to the technical field of molecular biological strain detection, in particular to a multiplex real-time fluorescence PCR detection primer composition for identifying streptococcus suis and pasteurella multocida, and a detection method using the detection primer composition.
Background
Swine streptococcosis (Suis-gdh) is an acute zoonosis caused by a plurality of serogroup streptococci, the epidemic situation is violent when the outbreak occurs, the spread is rapid, the body temperature of a sick pig is suddenly increased to be higher than 41 ℃, typical symptoms can not be seen almost, and the pig dies within a few hours. The swine streptococcosis is a second kind of animal epidemic disease specified in China, not only poses great threat to the world pig industry, but also endangers the safety of public health. Because the pathological changes of the disease are complex, the disease can be used as a preliminary diagnosis only according to the pathological changes and clinical data, and the diagnosis needs laboratory diagnosis. Clinical diagnosis is easily confused with swine fever, swine erysipelas and paratyphoid. Pathogens can be detected in nasal fluid, saliva, blood, muscle, viscera, and swollen joints of sick pig.
Porcine Pasteurella Multocida (PM) is an important zoonotic pathogen that can cause the development of infections in a variety of animals and humans. The swine plague with high mortality can be caused, is widely popularized in various countries in the world, and is frequently generated in large-scale pig farms in China. The prevalence of pasteurella multocida seriously harms the health of humans and animals, causing significant economic losses to the world's swine industry. At present, the traditional pathogen separation method is still commonly adopted in domestic veterinary clinic diagnosis of the disease, and the method is complex, time-consuming and low in sensitivity.
The key to successful control is to establish a method for researching and rapidly detecting streptococcus suis and pasteurella multocida. Since streptococcus suis and pasteurella multocida are common important pathogens of porcine respiratory disease syndrome and are often mixed infections, traditional diagnostic techniques such as bacterial isolation, immunological tests and the like are time-consuming and labor-consuming, are not suitable for clinical rapid diagnosis, and are not suitable for large-scale epidemiological investigation. With the rapid development of molecular biotechnology, many detection methods are established for nucleic acids of pathogenic microorganisms, including PCR detection technology, nucleic acid probe hybridization technology, loop-mediated isothermal amplification (LAMP), and the like. The PCR technology is one of important research means in molecular biology, and the method is time-saving, simple, convenient, economical and practical, and greatly improves the detection efficiency of clinical samples. On the basis of single PCR, a plurality of novel PCR technologies are established, such as multiplex PCR technology, real-time fluorescent quantitative PCR technology, multiplex fluorescent quantitative PCR technology, reverse transcription PCR technology and the like.
For example, CN 108315401A discloses a triple PCR primer, a method and a kit for detecting Streptococcus suis type 2, Pasteurella suis multocida and Haemophilus parasuis. Real-time fluorescence quantitative PCR is recognized as a qualitative leap in PCR diagnostic technology. In the prior art, a double fluorescence PCR detection method established by taking streptococcus suis and pasteurella suicidae as research objects does not exist. As the size of the amplified fragment of the ordinary PCR is generally 150-1000bp, the fluorescence PCR requires the content of the primer G + C to be 40-60% and the size of the amplified fragment is preferably 100-300bp, which has higher requirements on the fluorescence PCR, not all the genes of the ordinary multiplex PCR can be detected by using the multiplex fluorescence PCR, which brings new problems for the development of the multiplex fluorescence PCR detection technology.
Disclosure of Invention
In order to solve the problem that a double fluorescence PCR detection method which is established by taking streptococcus suis and pasteurella multocida as research objects does not exist in the prior art, the invention provides a fluorescence PCR detection primer composition for identifying streptococcus suis and pasteurella multocida.
The invention also provides a fluorescence PCR detection method for identifying streptococcus suis and pasteurella multocida. The method has high specificity and specificity, high amplification efficiency, high sensitivity, high accuracy, good reproducibility and short detection period, can detect the DNA amplification reaction in real time, and has high feasibility and application prospect.
The gdh gene is a newly discovered factor related to the virulence of streptococcus suis, belongs to a glutamate dehydrogenase protein family, is exposed on the cell wall of thalli, and has important significance on the pathogenicity of bacteria. The nucleotide sequence of gdh gene between different serotypes of streptococcus suis is highly conserved, the homology is about 96-100%, the protein antigen can be used as an important marker antigen for detecting the streptococcus suis, the infection of the streptococcus suis can be accurately detected, and the protein antigen has important significance for epidemiological research of the streptococcus suis.
The plpE gene exists in all serotypes of PM and is a specific conserved gene of the strain, and the sequence similarity of the plpE gene of different serotypes of PM is more than 92 percent, so that the ppE gene is suitable for detecting PM pathogens.
In order to achieve the purpose, the invention adopts the following technical scheme:
a multiplex fluorescence PCR detection primer composition for identifying streptococcus suis and pasteurella multocida comprises a streptococcus suis specific upstream and downstream primer and a specific probe, a pasteurella multocida specific upstream and downstream primer and a specific probe, and a bacteria universal upstream and downstream primer and a probe, wherein nucleotide sequences are respectively as follows:
streptococcus Suis specific upstream primer Suis-gdh QF: 5'-CCTCCGCCAGTTTGATGC-3', respectively;
specific downstream primer Suis-gdh QR 5'-GAAGGATTTACCGTTTGCTGC-3' of streptococcus Suis;
streptococcus Suis specific probe Suis-gdh QP:5 '-X1-TCATTGATCCGCCCAGAAGCA-Y1-3';
specific upstream primer PM QF: 5'-TAGTTGCATGTAGCGGTGGT-3' of swine pasteurella multocida;
specific downstream primers PM QR: 5'-AGGGGCTTGAAAGGAGGA-3' of swine pasteurella multocida;
the specific probe PM QP of the swine pasteurella multocida is 5 '-X2-CGCTGGAAATCGTGCTGACC-Y2-3';
bacterial universal upstream primer 16 SF: 5'-CGTATTACCGCGGCTGCTGG-3', respectively;
bacterial universal downstream primer 16 SR: 5'-GATTAGATACCCTGGTAGTCC-3', respectively;
bacterial universal probe 16 SP: 5 '-X3-CCGCCTTCGCCACCGGTGTTCTT-Y3-3'.
The 5 'end of the multiplex fluorescence PCR detection primer composition, the streptococcus suis specific probe, the pasteurella multocida and the bacteria universal probe is preferably X1, X2 and X3 which are respectively different from other two fluorescent modifications in FAM, JOE, CY5, ROX and CY3, and the 3' end of Y1, Y2 and Y3 which are respectively different from other two quenching groups in Dabcyl, BHQ1, BHQ2 and TAMRA.
The primer composition for multiplex fluorescence PCR detection is preferable
Specific probe for Streptococcus Suis Suis-gdh QP:5 '-TAMRA-TCATTGATCCGCCCAGAAGCA-BHQ 2-3';
specific probe PM QP of porcine pasteurella multocida: 5 '-JOE-CGCTGGAAATCGTGCTGACC-BHQ 2-3';
bacterial universal probe 16 SP: 5 '-CY 5-CCGCCTTCGCCACCGGTGTTCTT-BHQ 2-3'.
A multiplex fluorescence PCR detection kit for identifying Streptococcus suis and Pasteurella multocida, preferably comprising the multiplex fluorescence PCR detection primer composition of any one of claims 1 to 3.
The multiplex fluorescence PCR detection kit comprises a 20 mu L PCR amplification system: 2 XTaqMan Master Mix10 μ L, final concentrations of Suis-gdh QF, Suis-gdh QR, PM QF, PM QR, 16SF and 16SR were 0.25 μ M, respectively, final concentrations of Suis-gdh QP, PM QP and 16SP were 0.125 μ M, 2 μ L of 20 ng/. mu.L DNA template, and double distilled water made up to 20 μ L.
A multiplex fluorescence PCR detection method for identifying streptococcus suis and pasteurella multocida comprises the following steps:
1) extracting template DNA of a sample to be detected, and carrying out PCR amplification by using a reagent containing the multiplex fluorescence PCR detection primer composition in claim 1 or the multiplex fluorescence PCR detection kit in claim 2;
2) if the Suis-gdh QP and 16SP fluorescent modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is streptococcus Suis; if the PM QP and 16SP fluorescent modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is the swine pasteurella multocida; if the Suis-gdh QP and PM QP fluorescence modified probes have no amplification curve but 16SP has an amplification curve, if Ct is less than or equal to 35, the sample to be detected is not any one of the two bacteria.
The multiplex fluorescence PCR detection method preferably adopts an amplification program: at 95 ℃ for 2 min; 95 ℃ for 10 s; 58 ℃ for 35s, 40 cycles.
The multiplex fluorescence PCR detection method preferably performs detection on a fluorescence quantitative PCR instrument with 5 channels or more than 5 channels.
Furthermore, the invention also provides a genome-based accurate quantitative detection method for streptococcus suis and pasteurella multocida, which comprises the following specific steps:
preparation of streptococcus suis genome-standard: initial concentration was 30 ng/. mu.L, Streptococcus suis genome size was 2.007Mb, Copy number ═ 6.02X 1023)×(30ng/ul×10-9)/(2.007×106×660)＝1.36×107copies/μL；
Preparation of a swine pasteurella multocida-standard: an initial concentration of 30 ng-μ L, Haemophilus parasuis genome size 2.3Mb, Copy number ═ 6.02X 1023)×(30ng/ul×10-9)/(2.3×106×660)＝1.19×107copies/μL；
The genomic DNA of streptococcus suis and pasteurella multocida are respectively extracted from 1.36X 107copies/. mu.L and 1.19X 107The copies/. mu.L was diluted in 10X steps with 5 gradients, i.e. 106、105、104、103、102An order of magnitude copy number. And (3) performing real-time fluorescent quantitative PCR detection by taking 5 gradient standard products as templates to generate a standard curve, and simultaneously performing accurate quantification on the sample to be detected.
The invention has the beneficial effects that:
the streptococcus suis and the pasteurella multocida respectively take gdh and plpe as exclusive target genes, and have high specificity and specificity; the invention designs the universal primers and universal probes for bacteria, can effectively avoid false negative caused by PCR inhibition factors, and can simultaneously identify two bacteria by 3-color multiplex real-time fluorescence PCR. The invention can provide qualitative detection of streptococcus suis and pasteurella multocida, can realize accurate quantification, has high amplification efficiency, high sensitivity, high accuracy, good reproducibility and short detection period, can complete detection within 1.5 hours, can monitor in real time, has high feasibility and application prospect, and provides a scientific and reliable method for identifying pathogenic microorganisms and reducing economic loss of culture.
Drawings
FIG. 1 is a graph showing the amplification of Streptococcus suis under different fluorescent-modified probes; as can be seen from the figure: when the Suis-gdh QP and 16SP probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is streptococcus Suis;
FIG. 2 is a graph showing the amplification curves of porcine Pasteurella multocida with different fluorescence-modified probes; as can be seen from the figure: when the PM QP and 16SP probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is the swine pasteurella multocida;
FIG. 3 is a graph showing the amplification curves of bacteria other than two bacteria with different fluorescence-modified probes; as can be seen from the figure: when only the 16SP probe has an amplification curve, if Ct is less than or equal to 35, the sample to be detected is not one of the two bacteria;
FIG. 4 is a graph showing the sensitivity amplification curves of Streptococcus suis plates at 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng and 0.0001ng, respectively; as can be seen from the figure: the lowest detection limit is 0.01 ng;
FIG. 5 is a graph showing the sensitivity amplification curves of 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng and 0.0001ng of the porcine Pasteurella multocida template, respectively; as can be seen from the figure: the lowest detection limit is 0.01 ng;
FIG. 6 quasi-graph of porcine Pasteurella multocida;
FIG. 7 Standard graph of Streptococcus suis.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.
The experimental materials, reagents and instruments used in the present invention are as follows:
experimental materials: streptococcus suis, Pasteurella multocida, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Escherichia coli, Pseudomonas purulenta, Staphylococcus aureus, Bacillus subtilis, avian paragallinarum, Bacillus coagulans, Lactobacillus reuteri, Lactobacillus plantarum, Bacillus carlsbergii, and enterococcus faecium.
The reagents used were: the bacterial DNA extraction kit was purchased from Bao bioengineering (Dalian) Co., Ltd. The primers and probes were synthesized by Biotechnology engineering (Shanghai) Ltd. 2 × TaqMan Master Mix is DBI Bioscience brand. DNA sequencing was performed by the Biotechnology center, institute of agricultural sciences, Shandong province.
The apparatus used was: the ABI 7500 fluorescent quantitative PCR instrument is a product of ABI company, and the Takara PCR instrument is a product of Bao bioengineering (Dalian) company Limited. Model 5424D high speed centrifuge is a product of Eppendorf corporation.
Example 1
1. Extracting DNA of a streptococcus suis sample, a pasteurella multocida sample and other bacterial samples:
extracting by using a bacterial DNA extraction kit, wherein the specific operation steps are shown in the kit specification. The purity and concentration of the extracted genomic DNA are measured by an ultraviolet spectrophotometer. The measured OD260/OD280 values are all about 1.8-1.9, and the concentration is more than 10 ng/muL, which shows that the DNA has high purity and moderate concentration and meets the PCR amplification requirement.
2. Selection of target genes and design of primers: the nucleotide sequences of the primers and probes of streptococcus Suis (Suis) and Pasteurella Multocida (PM) with Gdh gene, plpEgene and OmlA gene as specific target genes are shown in table 1.
TABLE 1 nucleotide sequences of primers and probes
3. Preparing a standard product:
preparation of streptococcus suis genome-standard: initial concentration was 30 ng/. mu.L, Streptococcus suis genome size was 2.007Mb, Copy number ═ 6.02X 1023)×(30ng/ul×10-9)/(2.007×106×660)＝1.36×107copies/μL；
Preparation of a swine pasteurella multocida-standard: the initial concentration was 30 ng/. mu.L, the Haemophilus parasuis genome size was 2.3Mb, and the Copy number was (6.02X 10)23)×(30ng/ul×10-9)/(2.3×106×660)＝1.19×107copies/μL；
The genomic DNA of Streptococcus suis and Pasteurella multocida were diluted in 10X stepwise from 1.36X 107 copies/. mu.L and 1.19X 107 copies/. mu.L, respectively, in 5 gradients, i.e., 106、105、104、103、102An order of magnitude copy number. And (3) performing real-time fluorescent quantitative PCR detection by taking 5 gradient standard products as templates to generate a standard curve, and simultaneously performing accurate quantification on the sample to be detected.
4. Fluorescence detection:
preferably, 20. mu.L of real-time fluorescent PCR amplification system is used, and the reaction system is shown in Table 2.
TABLE 2 PCR reaction amplification System
5. The PCR amplification conditions were: 2min at 95 ℃; fluorescence signals were collected at 95 ℃ for 10s, 58 ℃ for 35s, 40 cycles.
6. And (4) analyzing results: and (3) setting a streptococcus suis positive control, a pasteurella multocida positive control, a negative control and a blank control in each test, opening analysis software after the test is finished, analyzing the test result, giving out delta Rn (fluorescence increase value in the nth cycle) and an amplification curve Ct value, and judging whether the sample to be detected is the pasteurella multocida or the streptococcus suis according to the probe fluorescence signal and the amplification curve Ct value. The result is shown in figure 1, when the Suis-gdh QP and 16SP probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is streptococcus Suis; FIG. 2 shows that when the PM QP and 16SP probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is the swine pasteurella multocida; FIG. 3 shows that when only 16SP probe has an amplification curve, and Ct ≦ 35 is satisfied, it indicates that the sample to be tested is not any of the two bacteria.
FIG. 6 is a graph showing the standard curve of Pasteurella multocida, and the copy number of Haemophilus parasuis in the sample to be examined is calculated by the equation y-3.547X +21.167(R2:0.997, Eff%: 91.38);
FIG. 7 is a standard curve diagram of Streptococcus suis, which is obtained by calculating the copy number of Streptococcus suis of the sample to be examined by the equation y-3.971X +25.296(R2:0.990, Eff%: 90.30).
Example 2 specificity verification
By using the primers and the probes designed by the invention, the real-time fluorescence PCR detection is carried out by taking the total genome DNA of streptococcus suis, pasteurella suis, haemophilus parasuis, actinobacillus pleuropneumoniae, escherichia coli, purulent bacillus, staphylococcus aureus, bacillus subtilis, avibacterium paragallinarum, bacillus coagulans, lactobacillus reuteri, lactobacillus plantarum, bacillus calfsii and enterococcus faecium as templates respectively, and the specificity of the primers and the probes is verified. The results are shown in Table 3 and FIG. 3, which show that the probe and primer designed in this study have strong specificity.
TABLE 3 specificity verification test
Example 3 sensitivity test
The genomic DNA of the streptococcus suis and the pasteurella multocida is quantified to 5 ng/mu L respectively, and diluted according to 10 multiplied by gradient, 2.0 mu L of each gradient is taken as a template amount (namely, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng, 0.0001ng and 0.00001ng) to carry out real-time fluorescence quantitative PCR detection, and the detection limit of the invention is evaluated. As shown in FIGS. 4-5, the sensitivities of Streptococcus suis and Pasteurella suis are both 0.01ng, and the results show that the quantitative detection limit of the method is 0.01ng, which indicates that the method provided by the invention has high sensitivity and higher sensitivity compared with the common PCR.
Example 4 clinical suspicious sample detection
The multiple real-time fluorescence PCR detection method for streptococcus suis and pasteurella multocida established by the invention detects 22 clinical suspicious samples, wherein the types of the samples comprise morbid materials and serum in different areas such as Shouguese tobacco station and the like. The source and number of 22 clinically suspect samples were tested simultaneously using viral isolation and sequencing. The fluorescence quantitative detection result is shown in table 4, and the result shows that the method established by the invention is completely consistent with the verification of the sequencing result after virus separation, and the method is accurate and reliable.
TABLE 4
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (5)
1. A multiplex fluorescence PCR detection primer probe group for identifying streptococcus suis and pasteurella multocida is characterized by comprising specific upstream and downstream primers and a specific probe of streptococcus suis, specific upstream and downstream primers and a specific probe of pasteurella multocida, and a universal upstream and downstream primer and a universal probe of bacteria, wherein the nucleotide sequences are respectively as follows:
streptococcus Suis specific upstream primer Suis-gdhQF: 5'-CCTCCGCCAGTTTGATGC-3', respectively;
specific downstream primer Suis-gdhQR 5'-GAAGGATTTACCGTTTGCTGC-3' of streptococcus Suis;
streptococcus Suis specific probe Suis-gdhQP: 5 '-X1-TCATTGATCCGCCCAGAAGCA-Y1-3';
specific upstream primer PMQF: 5'-TAGTTGCATGTAGCGGTGGT-3' of swine pasteurella multocida;
specific downstream primer PMQR: 5'-AGGGGCTTGAAAGGAGGA-3' of swine pasteurella multocida;
the specific probe PMQP of the swine pasteurella multocida is 5 '-X2-CGCTGGAAATCGTGCTGACC-Y2-3';
bacterial universal upstream primer 16 SF: 5'-CGTATTACCGCGGCTGCTGG-3', respectively;
bacterial universal downstream primer 16 SR: 5'-GATTAGATACCCTGGTAGTCC-3', respectively;
bacterial universal probe 16 SP: 5 '-X3-CCGCCTTCGCCACCGGTGTTCTT-Y3-3';
x1, X2 and X3 at the 5 'end of the probe are respectively fluorescence modifications different from the other two of FAM, JOE, CY5, ROX and CY3, and Y1, Y2 and Y3 at the 3' end of the probe are respectively different from the other two of Dabcyl, BHQ1, BHQ2 and TAMRA.
2. A multiplex fluorescence PCR detection kit for identifying Streptococcus suis and Pasteurella multocida, which is characterized by comprising the multiplex fluorescence PCR detection primer probe set according to claim 1.
3. The use of the multiplex fluorescent PCR detection primer probe set of claim 1 in the preparation of a product for identifying Streptococcus suis and Pasteurella multocida, wherein the product comprises the following use methods:
1) extracting template DNA of a sample to be detected, and performing PCR amplification by using the multiple fluorescence PCR detection primer probe group;
2) if the Suis-gdhQP and 16SP fluorescent modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is streptococcus Suis; if the PMQP and 16SP fluorescent modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is the swine pasteurella multocida; if the Suis-gdhQP fluorescence modified probe and the PMQP fluorescence modified probe have no amplification curve but 16SP has an amplification curve, if Ct is less than or equal to 35, the sample to be detected is not any one of the two bacteria.
4. The use of claim 3, wherein the PCR amplification procedure: at 95 ℃ for 2 min; 95 ℃ for 10 s; 58 ℃ for 35s, 40 cycles.
5. The use of claim 3, wherein the identification is performed on a 5-channel or higher fluorescent quantitative PCR instrument.
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| NL2024510A NL2024510B1 (en) | 2018-12-19 | 2019-12-19 | Multiplex real-time fluorescence pcr detection primer composition and detection method for identifying streptococcus suis and swine pasteurella multocida |
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